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  • Molecular Imaging of Infective Endocarditis With 6''-[18F]Fluoromaltotriose Positron Emission Tomography-Computed Tomography. Circulation Wardak, M., Gowrishankar, G., Zhao, X., Liu, Y., Chang, E., Namavari, M., Haywood, T., Gabr, M. T., Neofytou, E., Chour, T., Qin, X., Vilches-Moure, J. G., Hardy, J., Contag, C. H., McConnell, M. V., Wu, J. C., Gambhir, S. S. 2020; 141 (21): 1729–31

    View details for DOI 10.1161/CIRCULATIONAHA.119.043924

    View details for PubMedID 32453662

  • Maltotriose-based probes for fluorescence and photoacoustic imaging of bacterial infections. Nature communications Zlitni, A., Gowrishankar, G., Steinberg, I., Haywood, T., Sam Gambhir, S. 2020; 11 (1): 1250

    Abstract

    Currently, there are no non-invasive tools to accurately diagnose wound and surgical site infections before they become systemic or cause significant anatomical damage. Fluorescence and photoacoustic imaging are cost-effective imaging modalities that can be used to noninvasively diagnose bacterial infections when paired with a molecularly targeted infection imaging agent. Here, we develop a fluorescent derivative of maltotriose (Cy7-1-maltotriose), which is shown to be taken up in a variety of gram-positive and gram-negative bacterial strains in vitro. In vivo fluorescence and photoacoustic imaging studies highlight the ability of this probe to detect infection, assess infection burden, and visualize the effectiveness of antibiotic treatment in E. coli-induced myositis and a clinically relevant S. aureus wound infection murine model. In addition, we show that maltotriose is an ideal scaffold for infection imaging agents encompassing better pharmacokinetic properties and in vivo stability than other maltodextrins (e.g. maltohexose).

    View details for DOI 10.1038/s41467-020-14985-8

    View details for PubMedID 32144257

  • Engineered immune cells as highly sensitive cancer diagnostics NATURE BIOTECHNOLOGY Aalipour, A., Chuang, H., Murty, S., D'Souza, A. L., Park, S., Gulati, G. S., Patel, C. B., Beinat, C., Simonetta, F., Martinic, I., Gowrishankar, G., Robinson, E. R., Aalipour, E., Zhian, Z., Gambhir, S. S. 2019; 37 (5): 531-+
  • The characterization of 18F-hGTS13 for molecular imaging of xC- transporter activity with positron emission tomography. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Beinat, C., Gowrishankar, G., Shen, B., Alam, I. S., Robinson, E., Haywood, T., Patel, C. B., Azevedo, E. C., Castillo, J., Ilovich, O., Koglin, N., Schmitt-Willich, H., Berndt, M., Mueller, A., Zerna, M., Srinivasan, A., Gambhir, S. S. 2019

    Abstract

    Purpose: The aim of this study was development of an improved positron emission tomography (PET) radiotracer for measuring xC- activity with increased tumor uptake and reduced uptake in inflammatory cells compared to (S)-4-(3-18F-Fluoropropyl)-L-glutamic acid (18F-FSPG). Experimental design: A racemic glutamate derivative, 18F-hGTS13 was evaluated in cell culture and animal tumor models. 18F-hGTS13 was separated into C5-epimers and the corresponding 18F-hGTS13-isomer1 and 18F-hGTS13-isomer2 evaluated in H460 tumor bearing rats. Preliminary studies investigate the cellular uptake of 18F-hGTS13-isomer2 in multiple immune cell populations and states. Results:18F-hGTS13 demonstrated excellent H460 tumor visualization with high tumor-to-background ratios, confirmed by ex vivo biodistribution studies. Tumor associated radioactivity of 18F-hGTS13 (7.5±0.9%ID/g, n = 3) was significantly higher than with 18F-FSPG (4.6±0.7%ID/g, n = 3, P = 0.01). 18F-hGTS13-isomer2 exhibited excellent H460 tumor visualization (6.3±1.1%ID/g, n-3), and significantly reduced uptake in multiple immune cell populations relative to 18F-FSPG. 18F-hGTS13-isomer2 exhibited increased liver uptake relative to 18F-FSPG (4.6±0.8%ID/g vs. 0.7±0.01%ID/g) limiting its application in hepatocellular carcinoma. Conclusion:18F-hGTS13-isomer2 is a new PET radiotracer for molecular imaging of xC- activity which may provide information regarding tumor oxidation states. 18F-hGTS13-isomer2 has potential for clinical translation for imaging cancers of the thorax due to the low background signal in healthy tissue.

    View details for DOI 10.2967/jnumed.119.225870

    View details for PubMedID 31171595

  • Molecular imaging of bacterial infections: Overcoming the barriers to clinical translation. Science translational medicine Ordonez, A. A., Sellmyer, M. A., Gowrishankar, G., Ruiz-Bedoya, C. A., Tucker, E. W., Palestro, C. J., Hammoud, D. A., Jain, S. K. 2019; 11 (508)

    Abstract

    Clinical diagnostic tools requiring direct sample testing cannot be applied to infections deep within the body, and clinically available imaging tools lack specificity. New approaches are needed for early diagnosis and monitoring of bacterial infections and rapid detection of drug-resistant organisms. Molecular imaging allows for longitudinal, noninvasive assessments and can provide key information about infectious processes deep within the body.

    View details for DOI 10.1126/scitranslmed.aax8251

    View details for PubMedID 31484790

    View details for PubMedCentralID PMC6743081

  • Positron emission tomography reporter gene strategy for use in the central nervous system PNAS Haywood, T., Beinat, C., Gowrishankar, G., Patel, C. B., Alam, I. S., Murty, S., Gambhir, S. S. 2019

    View details for DOI 10.1073/pnas.1901645116

  • Assessment of tumor redox status through (S)-4-(3-[18F]fluoropropyl)-L-glutamic acid positron emission tomography imaging of system xc- activity. Cancer research McCormick, P. N., Greenwood, H. E., Glaser, M., Maddocks, O. D., Gendron, T., Sander, K., Gowrishankar, G., Hoehne, A., Zhang, T., Shuhendler, A. J., Lewis, D. Y., Berndt, M., Koglin, N., Lythgoe, M. F., Gambhir, S. S., Arstad, E., Witney, T. H. 2018

    Abstract

    The cell's endogenous antioxidant system is vital to maintenance of redox homeostasis. Despite its central role in normal and pathophysiology, no non-invasive tools exist to measure this system in patients. The cystine/glutamate antiporter system xc- maintains the balance between intracellular reactive oxygen species and antioxidant production through the provision of cystine, a key precursor in glutathione biosynthesis. Here we show that tumor cell retention of a system xc--specific positron emission tomography radiotracer, (S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), decreases in proportion to levels of oxidative stress following treatment with a range of redox-active compounds. The decrease in [18F]FSPG retention correlated with a depletion of intracellular cystine resulting from increased de novo glutathione biosynthesis, shown through [U-13C6, U-15N2]cystine isotopic tracing. In vivo, treatment with the chemotherapeutic doxorubicin decreased [18F]FSPG tumor uptake in a mouse model of ovarian cancer, coinciding with markers of oxidative stress but preceding tumor shrinkage and decreased glucose utilization. Having already been used in pilot clinical trials, [18F]FSPG PET could be rapidly translated to the clinic as an early redox indicator of tumor response to treatment.

    View details for PubMedID 30401715

  • Reply: 6"-18F-Fluoromaltotriose PET Evaluation in Escherichia-Coli-Induced Myositis: is there Uptake Saturation in Control? Journal of nuclear medicine : official publication, Society of Nuclear Medicine Wardak, M., Gowrishankar, G., Gambhir, S. S. 2018

    View details for PubMedID 29653976

  • [18F]FSPG-PET reveals increased cystine/glutamate antiporter (xc-) activity in a mouse model of multiple sclerosis. Journal of neuroinflammation Hoehne, A., James, M. L., Alam, I. S., Ronald, J. A., Schneider, B., D'Souza, A., Witney, T. H., Andrews, L. E., Cropper, H. C., Behera, D., Gowrishankar, G., Ding, Z., Wyss-Coray, T., Chin, F. T., Biswal, S., Gambhir, S. S. 2018; 15 (1): 55

    Abstract

    The cystine/glutamate antiporter (xc-) has been implicated in several neurological disorders and, specifically, in multiple sclerosis (MS) as a mediator of glutamate excitotoxicity and proinflammatory immune responses. We aimed to evaluate an xc-specific positron emission tomography (PET) radiotracer, (4S)-4-(3-[18F]fluoropropyl)-L-glutamate ([18F]FSPG), for its ability to allow non-invasive monitoring of xc- activity in a mouse model of MS.Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by subcutaneous injection of myelin oligodendrocyte glycoprotein (MOG35-55) peptide in complete Freund's adjuvant (CFA) followed by pertussis toxin. Control mice received CFA emulsion and pertussis toxin without MOG peptide, while a separate cohort of naïve mice received no treatment. PET studies were performed to investigate the kinetics and distribution of [18F]FSPG in naïve, control, pre-symptomatic, and symptomatic EAE mice, compared to 18F-fluorodeoxyglucose ([18F]FDG). After final PET scans, each mouse was perfused and radioactivity in dissected tissues was measured using a gamma counter. Central nervous system (CNS) tissues were further analyzed using ex vivo autoradiography or western blot. [18F]FSPG uptake in human monocytes, and T cells pre- and post-activation was investigated in vitro.[18F]FSPG was found to be more sensitive than [18F]FDG at detecting pathological changes in the spinal cord and brain of EAE mice. Even before clinical signs of disease, a small but significant increase in [18F]FSPG signal was observed in the spinal cord of EAE mice compared to controls. This increase in PET signal became more pronounced in symptomatic EAE mice and was confirmed by ex vivo biodistribution and autoradiography. Likewise, in the brain of symptomatic EAE mice, [18F]FSPG uptake was significantly higher than controls, with the largest changes observed in the cerebellum. Western blot analyses of CNS tissues revealed a significant correlation between light chain of xc- (xCT) protein levels, the subunit of xc- credited with its transporter activity, and [18F]FSPG-PET signal. In vitro [18F]FSPG uptake studies suggest that both activated monocytes and T cells contribute to the observed in vivo PET signal.These data highlight the promise of [18F]FSPG-PET as a technique to provide insights into neuroimmune interactions in MS and the in vivo role of xc- in the development and progression of this disease, thus warranting further investigation.

    View details for PubMedID 29471880

  • A novel synthesis of 6''-[18 F]-fluoromaltotriose as a PET tracer for imaging bacterial infection. Journal of labelled compounds & radiopharmaceuticals Namavari, M., Gowrishankar, G., Srinivasan, A., Gambhir, S. S. 2018

    Abstract

    The aim of this study was to develop a positron emission tomography (PET) tracer to visualize and monitor therapeutic response to bacterial infections. In our continued efforts to find maltose based PET tracers that can image bacterial infections, we have designed and prepared 6''-[18 F]fluoromaltotriose as a second generation PET imaging tracer targeting the maltodextrin transporter of bacteria. We have developed methods to synthesize 6''-deoxy-6''-[18 F]fluoro-α-D-glucopyranosyl-(1-4)-O-α-D-glucopyranosyl-(1-4)-O-D-glucopyranose (6''-[18 F]-fluoromaltotriose) as a bacterial infection PET imaging agent. 6''-[18 F]fluoromaltotriose was prepared from precursor, 2'',3'',4''-tri-O-acetyl-6''-O-nosyl-α-D-glucopyranosyl-(1-4)-O-2',3',6'-tri-O-acetyl-α-D-glucopyranosyl-(1-4)-1,2,3,6-tetra-O-acetyl-D-glucopyranose (per-O-acetyl-6''-O-nosyl-maltotriose 4). This method utilizes the reaction between precursor 4 and anhydrous [18 F]KF/Kryptofix 2.2.2 in Dimethylformamide (DMF) at 85o C for 10 minutes to yield per-O-acetyl-6''-deoxy-6-'' [18 F]-fluoromaltotriose (7). Successive acidic and basic hydrolysis of the acetyl protecting groups in 7 produced 6''-[18 F]fluoromaltotriose (8). Also, cold 6''- [19 F]fluoromaltotriose was prepared from per-O-acetyl-6''-hydroxymaltotriose via a DAST reaction followed by a basic hydrolysis. A successful synthesis of 6''-[18 F]-fluoromaltotriose has been accomplished in 8±1.2 % radiochemical yield (decay corrected). Total synthesis time was 120 min. Serum stability of 6''-[18 F]fluoromaltotriose at 37o C indicated that 6''-[18 F]-fluoromaltotriose remained intact up to 2 h. In conclusion, we have successfully synthesized 6''-[18 F]-fluoromaltotriose via direct fluorination of an appropriate precursor of a protected maltotriose.

    View details for PubMedID 29314161

  • A PET Imaging Strategy to Visualize Activated T Cells in Acute Graft-versus-Host Disease Elicited by Allogenic Hematopoietic Cell Transplant. Cancer research Ronald, J. A., Kim, B., Gowrishankar, G., Namavari, M., Alam, I. S., D'Souza, A., Nishikii, H., Chuang, H., Ilovich, O., Lin, C., Reeves, R., Shuhendler, A., Hoehne, A., Chan, C. T., Baker, J., Yaghoubi, S. S., VanBrocklin, H. F., Hawkins, R., Franc, B. L., Jivan, S., Slater, J. B., Verdin, E. F., Gao, K. T., Benjamin, J., Negrin, R., Gambhir, S. S. 2017; 77 (11): 2893-2902

    Abstract

    A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This affects patient prognosis as treatments are dramatically less effective at late disease stages. Here, we show that a novel PET radiotracer, 2'-deoxy-2'-[18F]fluoro-9-β-D-arabinofuranosylguanine ([18F]F-AraG), targeted toward two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications. Cancer Res; 77(11); 2893-902. ©2017 AACR.

    View details for DOI 10.1158/0008-5472.CAN-16-2953

    View details for PubMedID 28572504

  • F-Fluoromaltotriose: A Second Generation PET Tracer Targeting the Maltodextrin Transporter in Bacteria. Journal of nuclear medicine Gowrishankar, G., Hardy, J., Wardak, M., Namavari, M., Reeves, R., Neofytou, E., Srinivasan, A., Wu, J., Contag, C., Gambhir, S. 2017

    Abstract

    Purpose: 6"-(18)F-fluoromaltotriose is a novel positron emission tomography (PET) tracer that can potentially be used to image and localize most bacterial infections, much like 2-deoxy-2-(18)F-fluoro-D-glucose ((18)F-FDG) has been used to image and localize many cancers. However, unlike (18)F-FDG, 6"-(18)F-fluoromaltotriose is not taken up by inflammatory lesions and appears to be specific to bacterial infections by targeting the maltodextrin transporter that is expressed in most Gram-positive and Gram-negative strains of bacteria. Materials and Methods: 6"-(18)F-fluoromaltotriose was synthesized with high radiochemical purity and evaluated in several clinically relevant bacterial strains incultures in vitro and in living mice. Results: 6"-(18)F-fluoromaltotriose was taken up in both Gram-positive and Gram-negative bacterial strains. 6"-[(18)F]-fluoromaltotriose was also able to detect Pseudomonas aeruginosa in a clinically relevant mouse model of wound infection. The utility of 6"-(18)F-fluoromaltotriose to help monitor antibiotic therapies was also evaluated in rats. Conclusion: 6"-(18)F-fluoromaltotriose is a promising new tracer that has significant diagnostic utility, with the potential to change the clinical management of patients suffering from infectious diseases of bacterial origin.

    View details for DOI 10.2967/jnumed.117.191452

    View details for PubMedID 28490473

  • The Exosome Total Isolation Chip. ACS nano Liu, F., Vermesh, O., Mani, V., Ge, T. J., Madsen, S. J., Sabour, A., Hsu, E. C., Gowrishankar, G., Kanada, M., Jokerst, J. V., Sierra, R. G., Chang, E., Lau, K., Sridhar, K., Bermudez, A., Pitteri, S. J., Stoyanova, T., Sinclair, R., Nair, V. S., Gambhir, S. S., Demirci, U. 2017

    Abstract

    Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the device's broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 μL) of blood.

    View details for DOI 10.1021/acsnano.7b04878

    View details for PubMedID 29090896

  • Imaging B cells in a mouse model of multiple sclerosis using (64)Cu-Rituximab-PET. Journal of nuclear medicine : official publication, Society of Nuclear Medicine James, M. L., Hoehne, A., Mayer, A. T., Lechtenberg, K., Moreno, M., Gowrishankar, G., Ilovich, O., Natarajan, A., Johnson, E. M., Nguyen, J., Quach, L., Han, M., Buckwalter, M., Chandra, S., Gambhir, S. S. 2017

    Abstract

    B lymphocytes are a key pathological feature of multiple sclerosis (MS), and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to non-invasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here we evaluated (64)Cu-Rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using positron emission tomography (PET). Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of Myelin Oligodendrocyte Glycoprotein fragment1-125 (MOG1-125) emulsified in complete Freund's adjuvant. Control mice received complete Freund's adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19h following (64)Cu-Rituximab administration. Mice were perfused and sacrificed after final PET scan, and radioactivity in dissected tissues was measured with a gamma-counter. CNS tissues from these mice were immunostained to quantify B cells or further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice compared to controls at all evaluated time points (e.g., 1h post-injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 %ID/g, p<0.05). (64)Cu-Rituximab-PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice compared to 1.25±0.08 and 2.24±0.11%ID/g for controls, p<0.05 for all regions except striatum and thalamus at 1h post-injection. Similarly, ex vivo biodistribution results revealed notably higher (64)Cu-Rituximab uptake in brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased (64)Cu-Rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using (64)Cu-Rituximab-PET. Results from these studies warrant further investigation of (64)Cu-Rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell-targeted therapeutics en route to the clinic.

    View details for PubMedID 28687602

  • AshwaMAX and Withaferin A inhibits gliomas in cellular and murine orthotopic models JOURNAL OF NEURO-ONCOLOGY Chang, E., Pohling, C., Natarajan, A., Witney, T. H., Kaur, J., Xu, L., Gowrishankar, G., D'Souza, A. L., Murty, S., Schick, S., Chen, L., Wu, N., Khaw, P., Mischel, P., Abbasi, T., Usmani, S., Mallick, P., Gambhir, S. S. 2016; 126 (2): 253-264

    Abstract

    Glioblastoma multiforme (GBM) is an aggressive, malignant cancer Johnson and O'Neill (J Neurooncol 107: 359-364, 2012). An extract from the winter cherry plant (Withania somnifera ), AshwaMAX, is concentrated (4.3 %) for Withaferin A; a steroidal lactone that inhibits cancer cells Vanden Berghe et al. (Cancer Epidemiol Biomark Prev 23: 1985-1996, 2014). We hypothesized that AshwaMAX could treat GBM and that bioluminescence imaging (BLI) could track oral therapy in orthotopic murine models of glioblastoma. Human parietal-cortical glioblastoma cells (GBM2, GBM39) were isolated from primary tumors while U87-MG was obtained commercially. GBM2 was transduced with lentiviral vectors that express Green Fluorescent Protein (GFP)/firefly luciferase fusion proteins. Mutational, expression and proliferative status of GBMs were studied. Intracranial xenografts of glioblastomas were grown in the right frontal regions of female, nude mice (n = 3-5 per experiment). Tumor growth was followed through BLI. Neurosphere cultures (U87-MG, GBM2 and GBM39) were inhibited by AshwaMAX at IC50 of 1.4, 0.19 and 0.22 µM equivalent respectively and by Withaferin A with IC50 of 0.31, 0.28 and 0.25 µM respectively. Oral gavage, every other day, of AshwaMAX (40 mg/kg per day) significantly reduced bioluminescence signal (n = 3 mice, p < 0.02, four parameter non-linear regression analysis) in preclinical models. After 30 days of treatment, bioluminescent signal increased suggesting onset of resistance. BLI signal for control, vehicle-treated mice increased and then plateaued. Bioluminescent imaging revealed diffuse growth of GBM2 xenografts. With AshwaMAX, GBM neurospheres collapsed at nanomolar concentrations. Oral treatment studies on murine models confirmed that AshwaMAX is effective against orthotopic GBM. AshwaMAX is thus a promising candidate for future clinical translation in patients with GBM.

    View details for DOI 10.1007/s11060-015-1972-1

    View details for Web of Science ID 000368728300005

  • PET imaging of tumor glycolysis downstream of hexokinase through noninvasive measurement of pyruvate kinase M2. Science translational medicine Witney, T. H., James, M. L., Shen, B., Chang, E., Pohling, C., Arksey, N., Hoehne, A., Shuhendler, A., Park, J., Bodapati, D., Weber, J., Gowrishankar, G., Rao, J., Chin, F. T., Gambhir, S. S. 2015; 7 (310): 310ra169-?

    Abstract

    Cancer cells reprogram their metabolism to meet increased biosynthetic demands, commensurate with elevated rates of replication. Pyruvate kinase M2 (PKM2) catalyzes the final and rate-limiting step in tumor glycolysis, controlling the balance between energy production and the synthesis of metabolic precursors. We report here the synthesis and evaluation of a positron emission tomography (PET) radiotracer, [(11)C]DASA-23, that provides a direct noninvasive measure of PKM2 expression in preclinical models of glioblastoma multiforme (GBM). In vivo, orthotopic U87 and GBM39 patient-derived tumors were clearly delineated from the surrounding normal brain tissue by PET imaging, corresponding to exclusive tumor-associated PKM2 expression. In addition, systemic treatment of mice with the PKM2 activator TEPP-46 resulted in complete abrogation of the PET signal in intracranial GBM39 tumors. Together, these data provide the basis for the clinical evaluation of imaging agents that target this important gatekeeper of tumor glycolysis.

    View details for DOI 10.1126/scitranslmed.aac6117

    View details for PubMedID 26491079

  • Synthesis of [(18)F]-labelled Maltose Derivatives as PET Tracers for Imaging Bacterial Infection. Molecular imaging and biology Namavari, M., Gowrishankar, G., Hoehne, A., Jouannot, E., Gambhir, S. S. 2015; 17 (2): 168-176

    Abstract

    To develop novel positron emission tomography (PET) agents for visualization and therapy monitoring of bacterial infections.It is known that maltose and maltodextrins are energy sources for bacteria. Hence, (18)F-labelled maltose derivatives could be a valuable tool for imaging bacterial infections. We have developed methods to synthesize 4-O-(α-D-glucopyranosyl)-6-deoxy-6-[(18)F]fluoro-D-glucopyranoside (6-[(18)F]fluoromaltose) and 4-O-(α-D-glucopyranosyl)-1-deoxy-1-[(18)F]fluoro-D-glucopyranoside (1-[(18)F]fluoromaltose) as bacterial infection PET imaging agents. 6-[(18)F]fluoromaltose was prepared from precursor 1,2,3-tri-O-acetyl-4-O-(2',3',-di-O-acetyl-4',6'-benzylidene-α-D-glucopyranosyl)-6-deoxy-6-nosyl-D-glucopranoside (5). The synthesis involved the radio-fluorination of 5 followed by acidic and basic hydrolysis to give 6-[(18)F]fluoromaltose. In an analogous procedure, 1-[(18)F]fluoromaltose was synthesized from 2,3, 6-tri-O-acetyl-4-O-(2',3',4',6-tetra-O-acetyl-α-D-glucopyranosyl)-1-deoxy-1-O-triflyl-D-glucopranoside (9). Stability of 6-[(18)F]fluoromaltose in phosphate-buffered saline (PBS) and human and mouse serum at 37 °C was determined. Escherichia coli uptake of 6-[(18)F]fluoromaltose was examined.A reliable synthesis of 1- and 6-[(18)F]fluoromaltose has been accomplished with 4-6 and 5-8 % radiochemical yields, respectively (decay-corrected with 95 % radiochemical purity). 6-[(18)F]fluoromaltose was sufficiently stable over the time span needed for PET studies (∼96 % intact compound after 1-h and ∼65 % after 2-h incubation in serum). Bacterial uptake experiments indicated that E. coli transports 6-[(18)F]fluoromaltose. Competition assays showed that the uptake of 6-[(18)F]fluoromaltose was completely blocked by co-incubation with 1 mM of the natural substrate maltose.We have successfully synthesized 1- and 6-[(18)F]fluoromaltose via direct fluorination of appropriate protected maltose precursors. Bacterial uptake experiments in E. coli and stability studies suggest a possible application of 6-[(18)F]fluoromaltose as a new PET imaging agent for visualization and monitoring of bacterial infections.

    View details for DOI 10.1007/s11307-014-0793-5

    View details for PubMedID 25277604

  • Investigation of 6-[F-18]-Fluoromaltose as a Novel PET Tracer for Imaging Bacterial Infection PLOS ONE Gowrishankar, G., Namavari, M., Jouannot, E. B., Hoehne, A., Reeves, R., Hardy, J., Gambhir, S. S. 2014; 9 (9)

    Abstract

    Despite advances in the field of nuclear medicine, the imaging of bacterial infections has remained a challenge. The existing reagents suffer from poor sensitivity and specificity. In this study we investigate the potential of a novel PET (positron emission tomography) tracer that overcomes these limitations.6-[¹⁸F]-fluoromaltose was synthesized. Its behavior in vitro was evaluated in bacterial and mammalian cultures. Detailed pharmacokinetic and biodistribution profiles for the tracer were obtained from a murine model.6-[¹⁸F]-fluoromaltose is taken up by multiple strains of pathogenic bacteria. It is not taken up by mammalian cancer cell lines. 6-[¹⁸F]-fluoromaltose is retained in infected muscles in a murine model of bacterial myositis. It does not accumulate in inflamed tissue.We have shown that 6-[¹⁸F]-fluoromaltose can be used to image bacterial infection in vivo with high specificity. We believe that this class of agents will have a significant impact on the clinical management of patients.

    View details for DOI 10.1371/journal.pone.0107951

    View details for Web of Science ID 000343679800058

    View details for PubMedCentralID PMC4171493

  • Molecular Photoacoustic Imaging of Follicular Thyroid Carcinoma CLINICAL CANCER RESEARCH Levi, J., Kothapalli, S., Bohndiek, S., Yoon, J., Dragulescu-Andrasi, A., Nielsen, C., Tisma, A., Bodapati, S., Gowrishankar, G., Yan, X., Chan, C., Starcevic, D., Gambhir, S. S. 2013; 19 (6): 1494-1502

    Abstract

    To evaluate the potential of targeted photoacoustic imaging as a noninvasive method for detection of follicular thyroid carcinoma.We determined the presence and activity of two members of matrix metalloproteinase family (MMP), MMP-2 and MMP-9, suggested as biomarkers for malignant thyroid lesions, in FTC133 thyroid tumors subcutaneously implanted in nude mice. The imaging agent used to visualize tumors was MMP-activatable photoacoustic probe, Alexa750-CXeeeeXPLGLAGrrrrrXK-BHQ3. Cleavage of the MMP-activatable agent was imaged after intratumoral and intravenous injections in living mice optically, observing the increase in Alexa750 fluorescence, and photoacoustically, using a dual-wavelength imaging method.Active forms of both MMP-2 and MMP-9 enzymes were found in FTC133 tumor homogenates, with MMP-9 detected in greater amounts. The molecular imaging agent was determined to be activated by both enzymes in vitro, with MMP-9 being more efficient in this regard. Both optical and photoacoustic imaging showed significantly higher signal in tumors of mice injected with the active agent than in tumors injected with the control, nonactivatable, agent.With the combination of high spatial resolution and signal specificity, targeted photoacoustic imaging holds great promise as a noninvasive method for early diagnosis of follicular thyroid carcinomas.

    View details for DOI 10.1158/1078-0432.CCR-12-3061

    View details for Web of Science ID 000316188900021

    View details for PubMedID 23349314

    View details for PubMedCentralID PMC3602312

  • Positron Emission Tomography of Cu-64-DOTA-Rituximab in a Transgenic Mouse Model Expressing Human CD20 for Clinical Translation to Image NHL MOLECULAR IMAGING AND BIOLOGY Natarajan, A., Gowrishankar, G., Nielsen, C. H., Wang, S., Iagaru, A., Goris, M. L., Gambhir, S. S. 2012; 14 (5): 608-616

    Abstract

    This study aims to evaluate (64)Cu-DOTA-rituximab (PETRIT) in a preclinical transgenic mouse model expressing human CD20 for potential clinical translation.(64)Cu was chelated to DOTA-rituximab. Multiple radiolabeling, quality assurance, and imaging experiments were performed. The human CD20 antigen was expressed in B cells of transgenic mice (CD20TM). The mice groups studied were: (a) control (nude mice, n = 3) that received 7.4 MBq/dose, (b) with pre-dose (CD20TM, n = 6) received 2 mg/kg pre-dose of cold rituximab prior to PETRIT of 7.4 MBq/dose, and (c) without pre-dose (CD20TM, n = 6) PETRIT alone received 7.4 MBq/dose. Small animal PET was used to image mice at various time points (0, 1, 2, 4, 24, 48, and 72 h). The OLINDA/EXM software was used to determine the human equivalent dose for individual organs.PETRIT was obtained with a specific activity of 545 ± 38.91 MBq/nmole, radiochemical purity >95%, and immunoreactivity >75%. At 24 h, spleenic uptake of PETRIT%ID/g (mean ± STD) with and without pre-dose was 1.76 ± 0.43% and 16.5 ± 0.45%, respectively (P value = 0.01). Liver uptake with and without pre-dose was 0.41 ± 0.51% and 0.52 ± 0.17% (P value = 0.86), respectively. The human equivalents of highest dose organs with and without pre-dose are osteogenic cells at 30.8 ± 0.4 μSv/MBq and the spleen at 99 ± 4 μSv/MBq, respectively.PET imaging with PETRIT in huCD20 transgenic mice provided human dosimetry data for eventual applications in non-Hodgkins lymphoma patients.

    View details for DOI 10.1007/s11307-011-0537-8

    View details for Web of Science ID 000308819300011

    View details for PubMedID 22231277

  • GLUT 5 Is Not Over-Expressed in Breast Cancer Cells and Patient Breast Cancer Tissues PLOS ONE Gowrishankar, G., Zitzmann-Kolbe, S., Junutula, A., Reeves, R., Levi, J., Srinivasan, A., Bruus-Jensen, K., Cyr, J., Dinkelborg, L., Gambhir, S. S. 2011; 6 (11)

    Abstract

    F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.

    View details for DOI 10.1371/journal.pone.0026902

    View details for Web of Science ID 000297154900052

    View details for PubMedID 22073218

    View details for PubMedCentralID PMC3206880

  • Reproducibility study of [F-18]FPP(RGD)(2) uptake in murine models of human tumor xenografts EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Chang, E., Liu, S., Gowrishankar, G., Yaghoubi, S., Wedgeworth, J. P., Chin, F., Berndorff, D., Gekeler, V., Gambhir, S. S., Cheng, Z. 2011; 38 (4): 722-730

    Abstract

    An (18)F-labeled PEGylated arginine-glycine-aspartic acid (RGD) dimer {[(18)F]FPP(RGD)(2)} has been used to image tumor α(v)β(3) integrin levels in preclinical and clinical studies. Serial positron emission tomography (PET) studies may be useful for monitoring antiangiogenic therapy response or for drug screening; however, the reproducibility of serial scans has not been determined for this PET probe. The purpose of this study was to determine the reproducibility of the integrin α(v)β(3)-targeted PET probe, [(18)F]FPP(RGD)(2,) using small animal PET.Human HCT116 colon cancer xenografts were implanted into nude mice (n = 12) in the breast and scapular region and grown to mean diameters of 5-15 mm for approximately 2.5 weeks. A 3-min acquisition was performed on a small animal PET scanner approximately 1 h after administration of [(18)F]FPP(RGD)(2) (1.9-3.8 MBq, 50-100 μCi) via the tail vein. A second small animal PET scan was performed approximately 6 h later after reinjection of the probe to assess for reproducibility. Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean or maximum activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility.The coefficient of variation (mean±SD) for %ID(mean)/g and %ID(max)/g values between [(18)F]FPP(RGD)(2) small animal PET scans performed 6 h apart on the same day were 11.1 ± 7.6% and 10.4 ± 9.3%, respectively. The corresponding differences in %ID(mean)/g and %ID(max)/g values between scans were -0.025 ± 0.067 and -0.039 ± 0.426. Immunofluorescence studies revealed a direct relationship between extent of α(ν)β(3) integrin expression in tumors and tumor vasculature with level of tracer uptake. Mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, noting tumor volume, as well as making uniform: the time of imaging after injection and the ROI size. Reanalysis of ROI placement displayed variability for %ID(mean)/g of 6.6 ± 3.9% and 0.28 ± 0.12% for %ID(max)/g.[(18)F]FPP(RGD)(2) small animal PET mouse tumor xenograft studies are reproducible with relatively low variability.

    View details for DOI 10.1007/s00259-010-1672-1

    View details for Web of Science ID 000288255500015

    View details for PubMedID 21125268

  • Imaging Target mRNA and siRNA-Mediated Gene Silencing In Vivo with Ribozyme-Based Reporters CHEMBIOCHEM So, M., Gowrishankar, G., Hasegawa, S., Chung, J., Rao, J. 2008; 9 (16): 2682-2691

    Abstract

    Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.

    View details for DOI 10.1002/cbic.200800370

    View details for Web of Science ID 000261001900019

    View details for PubMedID 18972511

  • Visualizing RNA splicing in vivo MOLECULAR BIOSYSTEMS Gowrishankar, G., Rao, J. 2007; 3 (5): 301-307

    Abstract

    Ribozymes are RNA molecules capable of associating with other RNA molecules through base-pairing and catalyzing various reactions involving phosphate group transfer. Of particular interest to us is the well known ribozyme from Tetrahymena thermophila capable of catalyzing RNA splicing in eukaryotic systems, chiefly because of its potential use as a gene therapy agent. In this article we review the progress made towards visualizing the RNA splicing mediated by the Tetrahymena ribozyme in single living mammalian cells with the beta-lactamase reporter system and highlight the development made in imaging RNA splicing with the luciferase reporter system in living animals.

    View details for DOI 10.1039/b617574k

    View details for Web of Science ID 000246156700001

    View details for PubMedID 17460789

  • Detection of mRNA in mammalian cells with a split ribozyme reporter CHEMBIOCHEM Hasegawa, S., Gowrishankar, G., Rao, J. 2006; 7 (6): 925-928

    View details for DOI 10.1002/cbic.200600061

    View details for Web of Science ID 000238171400011

    View details for PubMedID 16671127

  • Inhibition of mRNA deadenylation and degradation by different types of cell stress. Biological chemistry Gowrishankar, G., Winzen, R., Dittrich-Breiholz, O., Redich, N., Kracht, M., Holtmann, H. 2006; 387 (3): 323–27

    Abstract

    We have previously observed rapid and strong inhibition of mRNA deadenylation and degradation in response to UV-B light [Gowrishankar et al., Biol. Chem. 386 (2005), pp. 1287-1293]. Expression analysis using a microarray for inflammatory genes showed that UV-B light induces stabilization of all short-lived mRNAs assayed. Stabilization was observed in HeLa cells, as well as in the keratinocyte line HaCaT. It affected constitutively expressed mRNA species, as well as species induced by the inflammatory cytokine IL-1. Many of the latter encode proteins involved in inflammation, suggesting that stress-induced inhibition of mRNA deadenylation contributes to changes in inflammatory gene expression. Deadenylation and degradation of tet-off-expressed mRNAs were also inhibited upon exposure to H2O2. However, scavengers of reactive oxygen species did not interfere with UV-B-induced inhibition of degradation, arguing against the involvement of UV-induced H2O2 in these effects of UV-B light. Heat shock and hyperosmolarity also inhibited mRNA deadenylation and degradation, whereas gamma-radiation did not. Thus, inhibition of mRNA deadenylation and degradation is a cellular response elicited by several but not all inducers of cell stress.

    View details for DOI 10.1515/BC.2006.043

    View details for PubMedID 16542155

  • Inhibition of mRNA deadenylation and degradation by ultraviolet light. Biological chemistry Gowrishankar, G., Winzen, R., Bollig, F., Ghebremedhin, B., Redich, N., Ritter, B., Resch, K., Kracht, M., Holtmann, H. 2005; 386 (12): 1287–93

    Abstract

    Post-transcriptional mechanisms contribute to the changes in gene expression induced by cell stress. The effect of UV-B light on mRNA degradation in HeLa cells was investigated using a transcriptional chase system to determine the decay kinetics of tet-off vector-derived mRNAs containing or lacking a destabilizing AU-rich element. Degradation of both mRNAs was strongly inhibited in cells exposed to UV-B light. Removal of the poly(A)-tail, considered a crucial step in mRNA degradation, was strikingly impaired. UV light also inhibited deadenylation and degradation of endogenous mRNA of the chemoattractant cytokine interleukin (IL)-8. Both effects occurred rapidly and independently of newly induced genes. Importantly, stabilization of IL-8 mRNA was accompanied by a strong increase in the duration of IL-8 protein formation. Furthermore, general inhibition of protein synthesis, a hallmark of the response to cell stress, required far higher doses of UV-B than inhibition of mRNA deadenylation and degradation. The difference in sensitivity of cells to these effects of UV-B light establishes a dose range in which mRNA stabilization can lead to dramatically enhanced expression of proteins derived from normally unstable mRNAs, such as those of inflammatory cytokines, growth factors and proto-oncogenes, and thereby have a major impact on the response to UV light.

    View details for DOI 10.1515/BC.2005.146

    View details for PubMedID 16336123

  • Distinct domains of AU-rich elements exert different functions in mRNA destabilization and stabilization by p38 mitogen-activated protein kinase or HuR. Molecular and cellular biology Winzen, R., Gowrishankar, G., Bollig, F., Redich, N., Resch, K., Holtmann, H. 2004; 24 (11): 4835–47

    Abstract

    AU-rich elements (AREs) control the expression of numerous genes by accelerating the decay of their mRNAs. Rapid decay and deadenylation of beta-globin mRNA containing AU-rich 3' untranslated regions of the chemoattractant cytokine interleukin-8 (IL-8) are strongly attenuated by activating the p38 mitogen-activated protein (MAP) kinase/MAP kinase-activated protein kinase 2 (MK2) pathway. Further evidence for a crucial role of the poly(A) tail is provided by the loss of destabilization and kinase-induced stabilization in ARE RNAs expressed as nonadenylated forms by introducing a histone stem-loop sequence. The minimal regulatory element in the IL-8 mRNA is located in a 60-nucleotide evolutionarily conserved sequence with a structurally and functionally bipartite character: a core domain with four AUUUA motifs and limited destabilizing function on its own and an auxiliary domain that markedly enhances destabilization exerted by the core domain and thus is essential for the rapid removal of RNA targets. A similar bipartite structure and function are observed for the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE. Stabilization in response to p38/MK2 activation is seen with the core domain alone and also after mutation of the AUUUA motifs in the complete IL-8 ARE. Stabilization by ARE binding protein HuR requires different sequence elements. Binding but no stabilization is observed with the IL-8 ARE. Responsiveness to HuR is gained by exchanging the auxiliary domain of the IL-8 ARE with that of GM-CSF or with a domain of the c-fos ARE, which results in even stronger responsiveness. These results show that distinct ARE domains differ in function with regard to destabilization, stabilization by p38/MK2 activation, and stabilization by HuR.

    View details for DOI 10.1128/MCB.24.11.4835-4847.2004

    View details for PubMedID 15143177

    View details for PubMedCentralID PMC416423

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